Antimicrobial compounds, compositions and methods of use thereof

ABSTRACT

Described herein are antimicrobial compounds identified via a high-throughput screen to identify compounds that produce anucleate cells in  E. coli  after cell division occurs. Compound 1 (5-nonyloxytryptamine) and its analogs are small molecule inhibitors of the nucleoid occlusion system and/or proteins that are responsible for maintaining the structure of the chromosome. The antimicrobial compounds are useful to treat bacterial infections as well as to inhibit bacterial growth.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application62/020,490 filed on Jul. 3, 2014, which is incorporated herein byreference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH & DEVELOPMENT

This invention was made with government support under OD008735 awardedby the National Institutes of Health. The government has certain rightsin the invention.

FIELD OF THE DISCLOSURE

The present disclosure is related to broad-spectrum antimicrobialcompounds, pharmaceutical compositions comprising the antimicrobialcompounds, and methods of treating bacterial infections with theantimicrobial compounds.

BACKGROUND

While the prevalence of multi-drug resistant pathogens continues torise, the rate at which new clinical antimicrobials are introduced hasdeclined significantly. In addition, the treatment of persistentinfections has been complicated by pathogen phenotypes. Bacteria thatgrow very slowly are often associated with prolonged infections, andthey are particularly tolerant to many of the clinically importantclasses of antibiotics that inhibit rapidly growing cells. For example,the β-lactam family of antibiotics inhibits enzymes involved in thesynthesis of peptidoglycan, and is thus most effective at targetingmicrobes that grow rapidly and continuously synthesize new cell wall.Relying on antibiotics that require fast metabolism and growth createslong-term problems, because dormant bacteria, as well as thoseassociated with biofilms and other multicellular structures, may surviveantibiotic treatments, become predisposed to developing drug resistance,and cause a relapse.

Previously, an assay was developed to detect specific inhibitors ofchromosome portioning in Escherichia coli. (Oyamada et al., “AnucleateCell Blue Assay: A Useful Tool for Identifying Novel Type IITopoisomerase Inhibitors,” Antimicrobial Agents and Chemotherapy, 50,pp. 348-350, (2006)). In the so-called anucleate cell blue assay,detection of anucleate cell production is used to screen for specificinhibitors of chromosome portioning. Compounds that inhibit either DNAgyrase or topoisomerase IV in vitro were identified and theantibacterial activity against certain drug-resistant Staphylococcusaureus strains was measured. The anucleate cell blue assay thus appearsto be a useful tool for identifying potential antibiotics.

What are needed are new broad-spectrum antimicrobial compounds,particularly antimicrobial compounds that inhibit chromosome segregationduring cell division and/or inhibit proteins responsible for maintainingthe structure of the chromosome.

BRIEF SUMMARY

In one aspect, included herein is an antimicrobial compound selectedfrom

Also included are pharmaceutical compositions including the compoundsand a pharmaceutically acceptable excipient.

Further included herein is an antimicrobial compound of Formula I, or apharmaceutically acceptable salt thereof, as well as methods of treatinga subject in need of treatment for a bacterial infection and methods ofinhibiting bacterial growth with the compounds,

wherein

R is a substituted or unsubstituted C₅-C₁₈ alkyl group, a substituted orunsubstituted C₅-C₁₈ alkenyl group, a substituted or unsubstitutedC₅-C₁₈ alkynyl group, a substituted or unsubstituted C₅-C₁₈ cycloalkylgroup, a substituted or unsubstituted C₆-C₁₈ cycloalkenyl group, asubstituted or unsubstituted C₈-C₁₈ cycloalkynyl group, a substituted orunsubstituted C₅-C₁₈ heteroalkyl group, a substituted or unsubstitutedC₅-C₁₈ heterocycloalkyl group, a substituted or unsubstituted C₇-C₃₀arylalkyl group, a substituted or unsubstituted C₇-C₃₀ aryloxyalkylgroup, or a substituted or unsubstituted C₇-C₃₀ arylthioalkyl group;

R₁ and R₂ are each independently hydrogen, a substituted orunsubstituted C₁-C₁₈ alkyl group, a substituted or unsubstituted C₃-C₁₈cycloalkyl group, a substituted or unsubstituted C₆-C₂₀ aryl group, or asubstituted or unsubstituted C₇-C₃₀ arylalkyl group;

R₃, R₄, R₅, and R₆ are each independently hydrogen, a halogen, a cyanogroup, a nitrile group, a sulfonate group, an amine group, a substitutedor unsubstituted C₁-C₁₈ alkyl group, a substituted or unsubstitutedC₁-C₁₈ alkoxy group, a substituted or unsubstituted C₁-C₁₈ alkylthiogroup, a substituted or unsubstituted C₃-C₁₈ cycloalkyl group, asubstituted or unsubstituted C₃-C₁₈ cycloalkoxy group, a substituted orunsubstituted C₃-C₁₈ cycloalkylthio group, a substituted orunsubstituted C₆-C₂₀ aryl group; a substituted or unsubstituted C₆-C₂₀aryloxy group, a substituted or unsubstituted C₆-C₂₀ arylthio group, ora substituted or unsubstituted C₇-C₃₀ arylalkyl group;

m is 0 or 1; and

n is an integer of 1 to 5.

Further included herein is an antimicrobial compound of Formula II,Formula III, Formula IV, or a pharmaceutically acceptable salt thereof,as well as methods of treating a subject in need of treatment for abacterial infection and methods of inhibiting bacterial growth with thecompounds,

wherein R is a substituted or unsubstituted C₅-C₁₈ alkyl group, asubstituted or unsubstituted C₅-C₁₈ alkenyl group, a substituted orunsubstituted C₅-C₁₈ alkynyl group, a substituted or unsubstitutedC₅-C₁₈ cycloalkyl group, a substituted or unsubstituted C₆-C₁₈cycloalkenyl group, a substituted or unsubstituted C₈-C₁₈ cycloalkynylgroup, a substituted or unsubstituted C₅-C₁₈ heteroalkyl group, asubstituted or unsubstituted C₅-C₁₈ heterocycloalkyl group, asubstituted or unsubstituted C₇-C₃₀ arylalkyl group, a substituted orunsubstituted C₇-C₃₀ aryloxyalkyl group, or a substituted orunsubstituted C₇-C₃₀ arylthioalkyl group. Also included are methods oftreating bacterial infections and methods of inhibiting bacterial growthwith the compounds of Formula I.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 A and B show Rabbit RBC hemolysis assays performed at variousconcentrations above and below the MIC of 1 against E. coli BW25113(MIC=6.2 μg/mL). 0.25% Triton-X is a positive control, Phosphatebuffered saline (PBS) is a negative control and 1% DMSO is a solventcontrol. Columns represent means and errors bars represent standarddeviations of 3 replicates. A) The absorbance of heme (lysed RBCs) after6 hours of incubation at 37° C. B) The absorbance of heme (lysed RBCs)after 17 hours of incubation at 37° C.

The above-described and other features will be appreciated andunderstood by those skilled in the art from the following detaileddescription, and appended claims.

DETAILED DESCRIPTION

The inventors of the present application developed a high-throughputassay and screened small molecule libraries to identify compounds thatproduce anucleate cells of the model bacterium Escherichia coli afterdivision occurs (i.e., cells that lack a copy of the chromosome), whichprevents replication. The goal was to identify small molecule inhibitorsof the nucleoid occlusion system (which synchronizes chromosomesegregation between the mother and daughter cell and cell division)and/or proteins that are responsible for maintaining the structure ofthe chromosome (i.e., structural maintenance of chromosome proteins).

Specifically, a screen of small molecule libraries identified5-nonyloxytryptamine (Compound 1, Table 1) as a potent hit in thisscreen; this compound has been reported as an anticancer drug and iscurrently under evaluation in animal studies for its anti-canceractivity. Antibiotic activity of Compound 1 does not appear to have beenreported previously. To confirm the activity of Compound 1, the compoundwas synthesized and the minimum inhibitory concentration (MIC) andminimum bacterial concentration (MBC) were determined against a broadpanel of human pathogens. As shown herein, Compound 1 has an MIC of 5-20μM against several pathogens; the only exception was Pseudomonasaeruginosa for which Compound 1 has an MIC of 80 μM, which is consistentwith the challenges of drug efflux in this organism. Compound 1 hasbroad-spectrum activity against Gram-positive and Gram-negativebacteria; in some organisms Compound 1 has bacteriostatic activity andin others Compound 1 is bactericidal.

Several analogs of Compound 1 (Compounds 7-12, Table 1) in which thealkyl group at the 5-hydroxy position of tryptamine was varied weresynthesized and tested. Several structurally related compounds(Compounds 2-6, Table 1) were also tested against the panel ofpathogens. The O-alkoxytryptamine analogs (Compounds 7-11, Table 1) aredifferentiated from other tryptamines (Compounds 2-6, Table 1) todemonstrate that the activity of compounds 1 and 7-11 is unique to theO-alkoxytryptamine family of compounds. Compounds 2-6 were largelyineffective as antibiotics (See Examples). Of the compounds synthesizedand tested, at least Compounds 8, 10, 11 and 12 appear to be novelcompounds. Compounds 9, 10 and 11 displayed MICs that are either thesame or 2-fold different from 1, thereby suggesting that these compoundsshare the same potency.

TABLE 1 Compound 1 and analogs

1

7

8

9

10

11

12

2

3

4

13

14

15

16

17

18

19

5

6

In one aspect, an antimicrobial compound or a pharmaceuticallyacceptable salt thereof has Formula I:

wherein

R is a substituted or unsubstituted C₅-C₁₈ alkyl group, a substituted orunsubstituted C₅-C₁₈ alkenyl group, a substituted or unsubstitutedC₅-C₁₈ alkynyl group, a substituted or unsubstituted C₅-C₁₈ cycloalkylgroup, a substituted or unsubstituted C₆-C₁₈ cycloalkenyl group, asubstituted or unsubstituted C₈-C₁₈ cycloalkynyl group, a substituted orunsubstituted C₅-C₁₈ heteroalkyl group, a substituted or unsubstitutedC₄-C₁₈ heterocycloalkyl group, a substituted or unsubstituted C₇-C₃₀arylalkyl group, a substituted or unsubstituted C₇-C₃₀ aryloxyalkylgroup, or a substituted or unsubstituted C₇-C₃₀ arylthioalkyl group;

R₁ and R₂ are each independently hydrogen, a substituted orunsubstituted C₁-C₁₈ alkyl group, a substituted or unsubstituted C₃-C₁₈cycloalkyl group, a substituted or unsubstituted C₆-C₂₀ aryl group, or asubstituted or unsubstituted C₇-C₃₀ arylalkyl group;

R₃, R₄, R₅, and R₆ are each independently hydrogen, a halogen, a cyanogroup, a nitrile group, a sulfonate group, an amine group, a substitutedor unsubstituted C₁-C₁₈ alkyl group, a substituted or unsubstitutedC₁-C₁₈ alkoxy group, a substituted or unsubstituted C₁-C₁₈ alkylthiogroup, a substituted or unsubstituted C₃-C₁₈ cycloalkyl group, asubstituted or unsubstituted C₃-C₁₈ cycloalkoxy group, a substituted orunsubstituted C₃-C₁₈ cycloalkylthio group, a substituted orunsubstituted C₆-C₂₀ aryl group; a substituted or unsubstituted C₆-C₂₀aryloxy group, a substituted or unsubstituted C₆-C₂₀ arylthio group, ora substituted or unsubstituted C₇-C₃₀ arylalkyl group;

m is 0 or 1; and

n is an integer of 1 to 5.

In another aspect, an antimicrobial compound has the following FormulaII, III, IV or a pharmaceutically acceptable salt thereof,

wherein R is a substituted or unsubstituted C₅-C₁₈ alkyl group, asubstituted or unsubstituted C₅-C₁₈ alkenyl group, a substituted orunsubstituted C₅-C₁₈ alkynyl group, a substituted or unsubstitutedC₄-C₁₈ cycloalkyl group, a substituted or unsubstituted C₅-C₁₈cycloalkenyl group, a substituted or unsubstituted C₈-C₁₈ cycloalkynylgroup, a substituted or unsubstituted C₅-C₁₈ heteroalkyl group, asubstituted or unsubstituted C₅-C₁₈ heterocycloalkyl group, asubstituted or unsubstituted C₇-C₃₀ arylalkyl group, a substituted orunsubstituted C₇-C₃₀ aryloxyalkyl group, or a substituted orunsubstituted C₇-C₃₀ arylthioalkyl group.

In a specific aspect, R is a substituted or unsubstituted C₅-C₁₈ alkylgroup, a substituted or unsubstituted C₅-C₁₈ alkenyl group, or asubstituted or unsubstituted C₅-C₁₈ alkynyl group. In one aspect, R issubstituted with one or more C₁-C₂₀ alkyl groups, C₆-C₂₀ aryl, and/orC₇-C₁₃ arylalkyl groups. In another aspect, R is substituted with one ormore C₁-C₄ alkyl groups or a C₆-C₁₀ aryl group.

In a more specific aspect, R is a linear, substituted or unsubstitutedC₅-C₁₈ alkyl group.

The term “substituted”, as used herein, means that any one or morehydrogens on the designated atom or group is replaced with a group atleast one group selected from a halide (F—, Cl—, Br—, I—), a hydroxyl, aC₁ to C₂₀ alkoxy, a cyano, a C₁ to C₂₀ alkyl, a C₂ to C₁₆ alkenyl, a C₂to C₁₆ alkynyl, a C₆ to C₂₀ aryl, a C₇ to C₁₃ arylalkyl, a C₇ to C₁₃aryloxyalkyl, a C₇ to C₁₃ arylthioalkyl, a C₁ to C₂₀ heteroalkyl, a C₃to C₂₀ cycloalkyl, and a C₅ to C₁₅ heterocycloalkyl, provided that thedesignated atom's normal valence is not exceeded. When a substituent isoxo (i.e., ═O), then 2 hydrogens on the carbon atom are replaced. Whenaromatic moieties are substituted by an oxo group, the aromatic ring isreplaced by the corresponding partially unsaturated ring. For example apyridyl group substituted by oxo is a pyridone. Combinations ofsubstituents and/or variables are permissible only if such combinationsresult in stable compounds or useful synthetic intermediates. A stablecompound or stable structure is meant to imply a compound that issufficiently robust to survive isolation from a reaction mixture, andsubsequent formulation into an effective therapeutic agent.

In a specific aspect, R is a C₄-C₁₈ alkyl group substituted with one ormore a C₁ to C₂₀ alkyl groups, C₆ to C₂₀ aryl, and/or C₇ to C₁₃arylalkyl groups. In another aspect, R is a C₄-C₁₈ alkyl groupsubstituted with one or more C₁ to C₄ alkyl groups or a C₆ to C₁₀ arylgroup.

As used herein, the term “alkyl” indicates a branched or straight-chainsaturated aliphatic hydrocarbon group, having the specified number ofcarbon atoms. Thus, the term C₁-C₈ alkyl as used herein includes alkylgroups having from 1 to about 8 carbon atoms. When C₀-C_(n) alkyl isused herein in conjunction with another group, for example,phenylC₀-C₄alkyl, the indicated group, in this case phenyl, is eitherdirectly bound by a single covalent bond (C₀), or attached by an alkylchain having the specified number of carbon atoms, in this case from 1to about 2 carbon atoms. Examples of alkyl include, but are not limitedto, methyl, ethyl, n-propyl, iso-propyl, n-butyl, t-butyl, n-pentyl, andsec-pentyl.

As used herein, the term “alkenyl” indicates a branched orstraight-chain hydrocarbon group including at least one carbon-carbondouble bond, and having the specified number of carbon atoms. Examplesof alkenyl include, but are not limited to, ethenyl, propenyl, including1-propenyl and 2-propenyl, and propanedienyl.

As used herein, the term “alkynyl” indicates a branched orstraight-chain hydrocarbon group including at least one carbon-carbontriple bond, and having the specified number of carbon atoms. Examplesof alkynyl include, but are not limited to, ethynyl and propynyl,including 1-propynyl and 2-propynyl.

As used herein, the term “aryl” indicates aromatic groups containingonly carbon in the aromatic ring or rings. Such aromatic groups may befurther substituted with carbon or non-carbon atoms or groups. Typicalaryl groups contain 1 to 3 separate, fused, or pendant rings and from 6to about 18 ring atoms, without heteroatoms as ring members. Whereindicated aryl groups may be substituted. Such substitution may includefusion to a 5 to 7-membered saturated cyclic group that optionallycontains 1 or 2 heteroatoms independently chosen from N, O, and S, toform, for example, a 3,4-methylenedioxy-phenyl group. Examples of arylinclude, but are not limited to, phenyl, naphthyl, including 1-naphthyland 2-naphthyl, and bi-phenyl.

As used herein, the term “arylalkyl” indicates an aryl group covalentlylinked to an alkyl group that is linked to a compound, and having thespecified number of carbon atoms. Examples of arylalkyl include, but arenot limited to, benzyl and phenethyl, including 1-phenethyl and2-phenethyl.

As used herein, the term “aryloxyalkyl” indicates an aryl groupcovalently linked to an alkyl group via oxygen, and the alkyl groupfurther linked to a compound, and having the specified number of carbonatoms. Examples of aryloxyalkyl include, but are not limited to,phenoxymethyl and phenoxyethyl, including 1-phenoxyethyl and2-phenoxyethyl.

As used herein, the term “arylthioalkyl” indicates an aryl groupcovalently linked to an alkyl group via sulfur, and the alkyl groupfurther linked to a compound, and having the specified number of carbonatoms. Examples of arylthioalkyl include, but are not limited to,phenylthiomethyl and phenylthioethyl, including 1-phenylthioethyl and2-phenylthioethyl.

As used herein, the term “cycloalkyl” indicates a group including atleast one saturated hydrocarbon ring, and having the specified number ofcarbon atoms. Examples of cycloalkyl include, but are not limited to,cyclopropyl, cyclopropylmethyl, cyclopropylethyl, cyclobutyl,cyclopentyl, or cyclohexyl. Cycloalkyl may include a bridged or cagedsaturated group such as norbornyl or adamantyl.

As used herein, the term “cycloalkenyl” indicates a group including atleast one hydrocarbon ring and at least one carbon-carbon double bond inthe ring, and having the specified number of carbon atoms. Examples ofcycloalkenyl include, but are not limited to, cyclopentenyl, including1-cyclopentenyl, 2-cyclopentenyl, and 3-cyclopentenyl.

As used herein, the term “cycloalkynyl” indicates a group including atleast one hydrocarbon ring and at least one carbon-carbon triple bond inthe ring, and having the specified number of carbon atoms. Examples ofcycloalkynyl include, but are not limited to, cyclooctynyl, including1-cyclooctynyl, 2-cyclooctynyl, 3-cyclooctynyl, and 4-cyclooctynyl.

As used herein, the term “heteroalkyl” indicates an alkyl groupincluding at least one heteroatom covalently bonded to one or morecarbon atoms of the alkyl group. Each heteroatom may each independentlybe chosen from nitrogen (N), oxygen (O), sulfur (S), and phosphorus (P).Examples of heteroalkyl include, but are not limited to, methoxymethyland methylthiomethyl.

As used herein, the term “heterocycloalkyl” indicates a group includinga saturated cyclic ring containing from 1 to about 5 heteroatoms chosenfrom nitrogen (N), oxygen (O), and sulfur (S), and phosphorus (P) withremaining ring atoms being carbon. Heterocycloalkyl groups may have from3 to about 8 ring atoms, and more typically have from 5 to 7 ring atoms.A C₂-C₇ heterocycloalkyl group contains from 2 to about 7 carbon ringatoms and at least one ring atom chosen from nitrogen (N), oxygen (O),sulfur (S), and phosphorus (P). Examples of heterocycloalkyl groupsinclude, but are not limited to, morpholinyl, piperazinyl, piperidinyl,and pyrrolidinyl groups.

A dash (“-”) that is not between two letters or symbols is used toindicate a point of attachment for a substituent. For example, —COOH isattached through the carbon atom.

In certain situations, the compounds of Formula I or II may possessasymmetry so that the compounds can exist in different stereoisomericforms. These compounds can be, for example, racemates or opticallyactive forms. For compounds with two or more asymmetric elements, thesecompounds can additionally be mixtures of diastereomers. For compoundshaving asymmetric centers, it should be understood that all of theoptical isomers and mixtures thereof are encompassed. In addition,compounds with carbon-carbon double bonds may occur in Z- and E-forms,with all isomeric forms of the compounds being included in the presentinvention. In these situations, the single enantiomers, i.e., opticallyactive forms, can be obtained by asymmetric synthesis, synthesis fromoptically pure precursors, or by resolution of the racemates. Resolutionof the racemates can also be accomplished, for example, by conventionalmethods such as crystallization in the presence of a resolving agent, orchromatography, using, for example a chiral LC or HPLC column.

“Pharmaceutically acceptable salts” includes derivatives of thedisclosed compounds wherein the parent compound is modified by making anacid or base salt thereof, and further refers to pharmaceuticallyacceptable solvates of such compounds and such salts. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids; and thelike. The pharmaceutically acceptable salts include the conventionalsalts and the quaternary ammonium salts of the parent compound formed,for example, from inorganic or organic acids. For example, conventionalacid salts include those derived from inorganic acids such ashydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric andthe like; and the salts prepared from organic acids such as acetic,propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric,ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, mesylic, esylic, besylic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isethionic, HOOC—(CH₂)_(n)—COOH where n is 0-4, andthe like. The pharmaceutically acceptable salts of can be synthesizedfrom a parent compound that contains a basic or acidic moiety byconventional chemical methods. Generally, such salts can be prepared byreacting free acid forms of these compounds with a stoichiometric amountof the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate,bicarbonate, or the like), or by reacting free base forms of thesecompounds with a stoichiometric amount of the appropriate acid. Suchreactions are typically carried out in water or in an organic solvent,or in a mixture of the two. Generally, non-aqueous media like ether,ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred,where practicable.

In one aspect, provided herein are methods of treating a subject in needof treatment for a bacterial infection, comprising administering to theindividual an antimicrobial compound or composition as described herein.The bacteria can be actively growing or in the stationary phase. In oneaspect, administration of an antimicrobial compound is topicaladministration. In another aspect, administration of an antimicrobialcompound is systemic administration such as oral administration.

The bacteria causing the infection can be Gram-negative, Gram-positive,or bacteria that are neither Gram-negative nor Gram-positive.Gram-negative bacteria include Escherichia coli, Pseudomonas aeruginosa,Candidatus liberibacter, Agrobacterium tumefaciens, Branhamellacatarrhalis, Citrobacter diversus, Enterobacter aerogenes, Klebsiellapneumoniae, Proteus mirabilis, Salmonella typhimurium, Neisseriameningitidis, Serratia marcescens, Shigella sonnei, Shigella boydii,Neisseria gonorrhoeae, Acinetobacter baumannii, Salmonella enteriditis,Fusobacterium nucleatum, Veillonella parvula, Bacteroides forsythus,Actinobacillus actinomycetemcomitans, Aggregatibacteractinomycetemcomitans, Porphyromonas gingivalis, Helicobacter pylori,Francisella tularensis, Yersinia pestis, Vibrio cholera, Shigellaboydii, Morganella morganii, Edwardsiella tarda, Campylobacter jejuni,and Haemophilus influenzae. In another embodiment, the bacteria areGram-positive bacteria. Gram-positive bacteria include species ofBacillus, Listeria, Staphylococcus, Streptococcus, Enterococcus,Corynebacterium, Propionibacterium and Clostridium. SpecificGram-positive bacteria include Staphylococcus aureus, Staphylococcusepidermidis, Enterococcus faecium, Enterococcus faecalis, Streptococcuspyogenes, Bacillus anthracis and Bacillus cereus. In a specificembodiment, the bacteria are one or more drug resistant bacteria.Bacteria that are neither Gram-negative nor Gram-positive includeBorrelia burgdorferi, Mycobacterium leprae, Mycobacterium tuberculosisand other Mycobacteria. Further included are bacteria such as Chlamydiaand Mycoplasma that do not have a cell wall. In certain aspects, thebacteria are resistant bacteria such as carbapenam-resistant bacteria,methicillin-resistant Staphylococcus aureus, vanccomycin-resistantEnterococci or multi-drug resistant Neisseria gonorrhoeae.

In another aspect, a method of inhibiting bacterial growth comprisescontacting the bacteria with an antimicrobial compound as describedherein. The bacteria can be actively growing or in the stationary phase.Methods of inhibiting bacteria include methods useful for treatment of asubject (human or veterinary) and also include methods useful forinhibiting bacteria outside of a subject, such as use for sterilizationand disinfection.

In one embodiment, the bacteria are in the form of a biofilm. A biofilmis a complex aggregate of microorganisms such as bacteria, wherein thecells adhere to each other on a surface. The cells in biofilms arephysiologically distinct from planktonic cells of the same organism,which are single cells that can float or swim in liquid medium. Biofilmsare involved in, for example, urinary tract infections, middle earinfections, dental plaques, gingivitis, coatings of contact lenses,cystic fibrosis, and infections of joint prostheses and heart valves.

The antimicrobial compounds and compositions may be administeredprophylactically, chronically, or acutely. For example, such compoundsmay be administered prophylactically to patients known to be prone tobacterial infections, or who are known to have been exposed topotentially infectious agents. The compounds may also be administeredprophylactically to patients suffering from other conditions, such asAIDS or other immune-system-suppressing conditions that render themsusceptible to opportunistic infections. In addition to the preventionof such infections, chronic administration of the antimicrobialcompounds will typically be indicated in treating refractory conditions,such as persistent infection by multiple drug-resistant strains ofbacteria. Acute administration of the antimicrobial compounds isindicated to treat, for example, those subjects presenting withclassical indications of bacterial infection.

As used herein, “contacting” means that a compound is provided such thatit is capable of making physical contact with another element, such as amicroorganism, a microbial culture or a substrate. In anotherembodiment, the term “contacting” means that the compound is introducedinto a subject receiving treatment, and the compound is allowed to comein contact in vivo. Thus, contacting can include administration of acompound, that is, introducing the compound into the body, such as intothe systemic circulation. Administration routes include but are notlimited to, rectal, oral, buccal, sublingual, pulmonary, transdermal,transmucosal, as well as subcutaneous, intraperitoneal, intravenous, andintramuscular injection.

Since the antimicrobial compounds are antibacterially active and inhibitbacterial growth, they are also of use in treating bacterialcontamination of a substrate, such as hospital instruments or worksurfaces. In order to treat a contaminated substrate, the compounds maybe applied to the site of such contamination in an amount sufficient toinhibit bacterial growth.

In certain embodiments, the compounds are administered to a patient orsubject. A “patient” or “subject”, used equivalently herein, meansmammals and non-mammals. “Mammals” means a member of the class Mammaliaincluding, but not limited to, humans, non-human primates such aschimpanzees and other apes and monkey species; farm animals such ascattle, horses, sheep, goats, and swine; domestic animals such asrabbits, dogs, and cats; laboratory animals including rodents, such asrats, mice, and guinea pigs; and the like. Examples of non-mammalsinclude, but are not limited to, birds, and the like. The term “subject”does not denote a particular age or sex.

The phrase “effective amount,” as used herein, means an amount of anagent, which is sufficient enough to significantly and positively modifysymptoms and/or conditions to be treated (e.g., provide a positiveclinical response). The effective amount of an active ingredient for usein a pharmaceutical composition will vary with the particular conditionbeing treated, the severity of the condition, the duration of thetreatment, the nature of concurrent therapy, the particular activeingredient(s) being employed, the particular pharmaceutically-acceptableexcipient(s)/carrier(s) utilized, and like factors within the knowledgeand expertise of the attending physician. In general, the use of theminimum dosage that is sufficient to provide effective therapy ispreferred. Patients may generally be monitored for therapeuticeffectiveness using assays suitable for the condition being treated orprevented, which will be familiar to those of ordinary skill in the art.

The phrase “inhibitory amount”, as used herein, means an amount of anagent (a compound or composition), which is sufficient to reduce thelevel or activity of bacterial infection to a statistically significantlesser value as compared to when the agent is not present.

The amount of compound effective for any indicated condition will, ofcourse, vary with the individual subject being treated and is ultimatelyat the discretion of the medical or veterinary practitioner. The factorsto be considered include the condition being treated, the route ofadministration, the nature of the formulation, the subject's bodyweight, surface area, age and general condition, and the particularcompound to be administered. In general, a suitable effective dose is inthe range of about 0.1 to about 500 mg/kg body weight per day,preferably in the range of about 5 to about 350 mg/kg per day. The totaldaily dose may be given as a single dose, multiple doses, e. g., two tosix times per day, or by intravenous infusion for a selected duration.Dosages above or below the range cited above may be administered to theindividual patient if desired and necessary.

Also included herein are pharmaceutical compositions comprising theantimicrobial compounds. As used herein, “pharmaceutical composition”means a therapeutically effective amount of the compound together with apharmaceutically acceptable excipient, such as a diluent, preservative,solubilizer, emulsifier, adjuvant, and the like. As used herein“pharmaceutically acceptable excipients” are well known to those skilledin the art. In one aspect, a pharmaceutical composition is suitable fortopical administration. In another aspect, a pharmaceutical compositionis suitable for systemic administration.

Tablets and capsules for oral administration may be in unit dose form,and may contain excipients such as binding agents, for example syrup,acacia, gelatin, sorbitol, tragacanth, or polyvinyl-pyrrolidone; fillersfor example lactose, sugar, maize-starch, calcium phosphate, sorbitol orglycine; tabletting lubricant, for example magnesium stearate, talc,polyethylene glycol or silica; disintegrants for example potato starch,or acceptable wetting agents such as sodium lauryl sulfate. The tabletsmay be coated according to methods well known in normal pharmaceuticalpractice. Oral liquid preparations may be in the form of, for example,aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, ormay be presented as a dry product for reconstitution with water or othersuitable vehicle before use. Such liquid preparations may containconventional additives such as suspending agents, for example sorbitol,syrup, methyl cellulose, glucose syrup, gelatin hydrogenated ediblefats; emulsifying agents, for example lecithin, sorbitan monooleate, oracacia; non-aqueous vehicles (which may include edible oils), forexample almond oil, fractionated coconut oil, oily esters such asglycerine, propylene glycol, or ethyl alcohol; preservatives, forexample methyl or propyl p-hydroxybenzoate or sorbic acid, and ifdesired conventional flavoring or coloring agents.

For topical application to the skin, the drug may be made up into acream, lotion or ointment. Cream or ointment formulations that may beused for the drug are conventional formulations well known in the art.Topical administration includes transdermal formulations such aspatches.

For topical application to the eye, the inhibitor may be made up into asolution or suspension in a suitable sterile aqueous or non-aqueousvehicle. Additives, for instance buffers such as sodium metabisulphiteor disodium edeate; preservatives including bactericidal and fungicidalagents such as phenyl mercuric acetate or nitrate, benzalkonium chlorideor chlorhexidine, and thickening agents such as hypromellose may also beincluded.

The active ingredient may also be administered parenterally in a sterilemedium, either subcutaneously, or intravenously, or intramuscularly, orintrasternally, or by infusion techniques, in the form of sterileinjectable aqueous or oleaginous suspensions. Depending on the vehicleand concentration used, the drug can either be suspended or dissolved inthe vehicle. Advantageously, adjuvants such as a local anaesthetic,preservative and buffering agents can be dissolved in the vehicle.

Pharmaceutical compositions may conveniently be presented in unit dosageform and may be prepared by any of the methods well known in the art ofpharmacy. The term “unit dosage” or “unit dose” means a predeterminedamount of the active ingredient sufficient to be effective for treatingan indicated activity or condition. Making each type of pharmaceuticalcomposition includes the step of bringing the active compound intoassociation with a carrier and one or more optional accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing the active compound into association with a liquidor solid carrier and then, if necessary, shaping the product into thedesired unit dosage form.

The antimicrobial compounds may also be administered in combination withan additional active agent, such as, for example, an inhibitor ofbacterial efflux. Efflux pumps are proteins that unidirectionally removeantibiotics from cytoplasmic compartments, and are considered to be amechanism of antibacterial resistance. Bacterial efflux inhibitorsinclude chalcone compounds as disclosed in WO 11/075136, the polybasiccompounds disclosed in WO 10/054102, the quaternary alkyl ammoniumfunctional compounds disclosed in WO 08/141012, the compounds disclosedin WO 05/007162, the substituted polyamines of WO 04/062674, which areincorporated herein by reference in their entirety.

In another embodiment, the antimicrobial compounds of Formula I can beadministered with a second antibiotic. Exemplary second antibioticsinclude, for example, glycopeptides (e.g, vancomycin or teicoplanin);penicillins, such as amdinocillin, ampicillin, amoxicillin, azlocillin,bacampicillin, benzathine penicillin G, carbenicillin, cloxacillin,cyclacillin, dicloxacillin, methicillin, mezlocillin, nafcillin,oxacillin, penicillin G, penicillin V, piperacillin, and ticarcillin;cephalosporins, such as cefadroxil, cefazolin, cephalexin, cephalothin,cephapirin, cephradine, cefaclor, cefamandole, cefonicid, ceforanide,cefoxitin, and cefuroxime, cefoperazone, cefotaxime, cefotetan,ceftazidime, ceftizoxime, ceftriaxone, and moxalactam; carbapenems suchas imipenem; monobactams such as aztreonam; tetracyclines such asdemeclocycline, tigilcycline, doxycycline, methacycline, minocycline,and oxytetracycline; aminoglycosides such as amikacin, gentamicin,kanamycin, neomycin, netilmicin, paromomycin, spectinomycin,streptomycin, and tobramycin; polymyxins such as colistin,colistimathate, and polymyxin B, and erythromycins and lincomycins andalso sulfonamides such as sulfacytine, sulfadiazine, sulfisoxazole,sulfamethoxazole, sulfamethizole, and sulfapyridine; trimethoprim,quinolones, novobiocin, pyrimethamine, rifampin, quinolines,fluoroquinolines; and combinations thereof.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Materials and Methods

In Vivo Screen:

The high throughput screen for the anucleate cell assay was based on theassay described in Oyamada et al., “Anucleate Cell Blue Assay: A UsefulTool for Identifying Novel Type II Topoisomerase Inhibitors,”Antimicrobial Agents and Chemotherapy, 50, pp. 348-350, (2006). Briefly,E. coli anucleate cell strain (SH3210; ΔtrpR5 his λ pXX747) is used inthe assay. The plasmid pXX747 includes the repA gene under control ofthe λP_(R) promoter and LacZ gene. In nucleated cells, repressorssupplied by the chromosomal cI gene of λ phage and lacI gene keepexpression of repA and lacZ genes low. In anucleated cells, however,expression of repA is induced, causing plasmid amplification, andinduction of lacZ expression. The anucleated cells thus appearfluorescent in the presence of a fluorescent substrate (e.g., DDOAG) forLacZ. The protocol is summarized as follows:

-   -   1. Prepare overnight culture of E. coli anucleate cell strain        (SH3210) from LB-ampicillin plates.    -   2. Dilute starter culture 1:1000 with liquid LB treated with 50        mg/ml of ampicillin and incubate for approximately 2.5 hr at        37° C. until approximately 0.1 OD₆₀₀.    -   3. Split diluted culture into wells (200 μL)        -   i. Positive control: 1 μL of 5.0 mM rifampicin (final            2×MIC=25 μM)        -   ii. Negative control: 1 μL of DMSO    -   4. Incubate diluted culture with compounds for approximately 2.5        hr at 42° C. at 300 rpm in a clear plate.    -   5. Incubate cultures on ice for approximately 10 min until        condensation on the cover disappears.    -   6. Measure absorbance at a wavelength of 595 nm    -   7. In a black 96-well plate, add 80 μL of Z-buffer (600 mM        Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, 0.1 MgSO₄) to each well        pre-loaded with the following reagents:        -   a. Cell lysis solution called Popculture™ (1:5 Popculture™            to buffer). Popculture™ is a buffered mixture of            concentrated detergents.        -   b. 15 μM of 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl)            β-d-galactopyranoside (DDAOG) (3 μL of 5 mM DDAOG per 1 mL            of buffer)    -   8. Transfer 20 μL of culture to wells containing Z-buffer.    -   9. Incubate covered at room temperature for 15 min.    -   10. Incubate plate at 37° C. for 20 min.    -   11. Add 50 μL of 1 M Na₂CO₃ to each well.    -   12. Shake for 30 s and measure fluorescence at a wavelength of        659 nm.    -   13. Normalize the fluorescence measurements by dividing by        OD₅₉₅.

${{Beta}\text{-}{gal}\mspace{14mu}{units}^{*}} = \frac{{Fluorescence}_{659}}{{OD}_{595}}$

Bacterial Strains and Growth Conditions.

Organisms and strains used in this study and their genotypes aresummarized in Table 2. Luria-Bertani (LB) media (10 g/L tryptone, 10 g/LNaCl, 5 g/L yeast extract, pH 7.0) was used to grow all bacterialstrains, except Streptococcus pyogenes. Streptococcus pyogenes was grownin Mueller-Hinton broth containing 5% (v/v) sheep blood. All bacterialcultures were grown at 37° C. All the E. coli strains were grown whileshaking at 200 rpm, while clinical pathogens were incubated withoutshaking.

TABLE 2 List of Strains Organism/Strain Genotype/Description S. aureussea⁺ (Tmm^(s) Hem^(+a) Em^(s)) FRI 100 Streptococcus pyrogenes E. coliBW25113 Δ(araD-araB)567 ΔlacZ4787(::rrnB-3) lambda⁻ rph-1Δ(rhaD-rhaB)568 hsdR514 E. coli BW25113 BW25113 tolC::kan^(R) ΔtolC P.aeruginosa Prototroph PAO1 P. aeruginosa ilv-220 thr-9001 leu-9001met-9011 pur-67 aphA K1115 ΔmexCD-oprJ ΔmexAB-oprM P. aeruginosa PAO1ΔmexAB-oprM K1119 Salmonella Clinical isolates from the Department ofMedical typhimurium Microbiology and Immunology (MMI) at the Universityof Wisconsin-Madison Vibrio cholerae Clinical isolates from theDepartment of Medical Microbiology and Immunology (MMI) at theUniversity of Wisconsin-Madison Shigella boydii Clinical isolates fromthe Department of Medical Microbiology and Immunology (MMI) at theUniversity of Wisconsin-Madison Morganella Clinical isolates from theDepartment of Medical morganii Microbiology and Immunology (MMI) at theUniversity of Wisconsin-Madison Edwardsiella Clinical isolates from theDepartment of Medical tarda Microbiology and Immunology (MMI) at theUniversity of Wisconsin-Madison Acinetobacter Clinical isolates from theDepartment of Medical baumannii Microbiology and Immunology (MMI) at theUniversity of Wisconsin-Madison Enterobacter Clinical isolates from theDepartment of Medical aerogenes Microbiology and Immunology (MMI) at theUniversity of Wisconsin-Madison Klebsiella Clinical isolates from theDepartment of Medical pneumoniae Microbiology and Immunology (MMI) atthe University of Wisconsin-Madison

Determination of the Minimum Inhibitory Concentration (MIC) of BacterialGrowth.

The MIC of all the strains was determined in LB media exceptStreptococcus pyogenes, which was grown in Mueller-Hinton mediacontaining 5% (v/v) sheep blood. The micro-dilution method was utilizedin 96-well clear flat-bottom plates (100 μL/well) according to the CLSIguidelines. All strains were grown at 37° C. for 16-18 hours in presenceof various concentrations of compound. The MIC was determined by avisual inspection and experiments were performed in triplicate.

Calculation of the Minimum Bacterial Concentration (MBC).

The antibacterial mode (i.e., bactericidal, bacteriostatic) wasdetermined by calculating the quotient of the MBC and the MIC. Compoundswith a quotient >4 are defined as bacteriostatic; if the quotient islower than 4, compounds are defined as bactericidal. A micro-dilutionprotocol was used to perform MIC and MBC experiments in triplicateaccording to the CLSI guidelines. MIC endpoints were determined byidentifying the lowest concentration of antibiotic that completelyinhibited growth by visual inspection. The total volume (100 μL) in thewells with no visual growth were plated on 1.5% (w/v) LB agar or 1.5%(w/v) Mueller-Hinton agar with 5% sheep blood and incubated for 22 hoursat 37° C. The MBC was determined to be the lowest concentration ofantibiotic that did not produce visible colonies.

Example 1 Selection of Compounds

Small molecule libraries at the UW-Madison Small Molecular Screening andMedicinal Chemistry Facility that included the Prestwick library ofoff-patent, FDA-approved drugs and therapeutic compounds undergoingadvanced toxicological evaluation in animal models were screened usingthe high throughput anucleate cell assay. 5-nonyloxytryptamine (Compound1, Table 1) as a potent hit in this screen as it displayed a minimuminhibitory concentrations against E. coli BW25113 that was <100 μg/mL.

Compound 1 is known to be a serotonin receptor agonist; specifically, ittargets the 5-HT1Dβ human homolog of the serotonin receptor family.Compound 1 was initially discovered in experiments designed to identifya serotonin agonist that binds 5-HT1Dβ receptors selectively withoutinhibiting 5-HT1A receptors. The selectivity of inhibition is criticalas inhibition of the 5-HT1A receptors is believed to contribute to theside effects of the medically relevant serotonin agonist, sumatriptan.Sumatriptan displays 60-fold selectivity for 5-HT1Dβ receptors versus5-HT1A receptors; Compound 1 binds the 5-HT1Dβ with 260-foldselectivity, which makes it a promising serotonin receptor agonist forcancer treatment.

Compound 1 was screened against a broad panel of human pathogens (Table3). Compound 1 has an MIC of 3-12 μg/mL against the pathogens we tested;the only exception was Pseudomonas aeruginosa for which Compound 1 hasan MIC of 24 μg/mL, which is consistent with the challenges of drugefflux in this organism.

TABLE 3 Minimum inhibitory concentration (MIC) and minimum bacterialconcentration (MBC) of Compound 1 against various pathogenic bacterialstrains MBC MIC Antibacterial Bacterial Strain (μg/mL) (μg/mL) MBC/MICMode Pseudomonas 96 24 4 Bacteriostatic aeruginosa Salmonella 24 6 4Bacteriostatic typhimurium Vibrio cholerae 24 6 4 BacteriostaticShigella boydii 6 6 1 Bactericidal Klebsiella 6 6 1 Bactericidalpneumoniae Enterobacter >96 6 >4 Bacteriostatic aerogenes Acinetobacter6 6 1 Bactericidal baumannii Edwardsiella 12 6 2 Bactericidal tardaMorganella 24 12 2 Bactericidal morganii Staphylococcus 6 3 2Bactericidal aureus Streptococcus 24 24 1 Bactericidal pyogenes

Example 2 Synthesis of Compound 1 and its Analogs

5-nonyloxytryptamine was synthesized according to methods in the art,specifically the methods of Glennon et al., “Binding of O-AlkylDerivatives of Serotonin at Human 5-HT1Dβ Receptors,” J. Med Chem, 39,pp. 314-322, (1996).

N-Boc-Serotonin:

In a round bottom flask, potassium carbonate (1.5 equiv.) was added inone portion to a suspension of serotonin HCl (1 equiv.) in H₂O. Oncefully dissolved, di-tert-butyl dicarbonate (1 equiv.) was added viasyringe and the mixture was stirred at 25° C. for 24 hours. The reactionmixture was diluted with H₂O and the product was extracted three timeswith EtOAc. The combined EtOAc layers were washed with H₂O, 5% HCl andbrine. The EtOAc portion was dried with Mg₂SO₄ and evaporated to yield agreen/brown oil.

N-Boc-5-Alkyoxytryptamine:

In a round bottom flask, potassium carbonate (1.78 equiv.) was added toa solution of N-Boc-serotonin (1 equiv.) in acetonitrile. To thereaction suspension, bromoalkane (1 equiv.) was added via syringe andthe reaction mixture was stirred under argon at 25° C. for 24 hours.After the reaction mixture cooled to room temperature, the solid wasremoved by vacuum filtration and the solvent was evaporated underreduced pressure. The crude product was purified using flashchromatography with silica gel and 25% EtOAc/Hexane as the mobile phaseto yield N-Boc-5-alkyoxytryptamine as a yellow oil.

5-Alkyoxytryptamine:

A solution of HCl (3M) in EtOAc was added to a solution ofN-boc-5-alkyoxytryptamine in EtOAc and the reaction mixture was stirredvigorously at 25° C. for 2 hours. The solvent was evaporated underreduced pressure and the crude product was washed with anhydrous diethylether. The solid product was collected by vacuum filtration, washedthree times with anhydrous diethylether and once with EtOAc to yield theHCl salt of 5-alkyoxytryptamine.

Example 3 Measurement of Minimum Inhibitory Concentrations for Compound1 and its Analogs

Several analogs of Compound 1 (Compounds 7-12, Table 1) in which thealkyl group at the 5-hydroxy position of tryptamine and severalstructurally related compounds (Compounds 2-6, Table 1) were testedagainst the panel of pathogens (results are shown in Table 4).Comparison of the O-alkoxytryptamine analogs (Compounds 1 and 7-11) andother tryptamines (Compounds 2-6) demonstrate that the activity ofCompounds 1 and 7-11 is unique to the O-alkoxytryptamine family ofcompounds. Compounds 2-6 include tryptamine (the scaffold forneurotransmitters) and commercially available analogs of serotonin,including: 5-methoxytryptamine (full agonist towards all 5-HT receptorsexcept 5-HT3), 2-methyl-5-hydroxytryptamine (specific agonist activityat the 5-HT3 receptor), and 6-methoxytryptamine (an inactive agonist atthe 5-HT receptors, but structurally related to the others). Compounds2-6 were largely ineffective as antibiotics; data for Compounds 7-11against E. coli is summarized in Table 5.

Compounds 10 and 11 displayed MICs that are either the same or 2×different from Compound 1, thereby suggesting that these compounds sharethe same potency.

TABLE 4 Minimum inhibitory concentrations (MIC) of analogs with variouscarbon chain lengths and substituents at the 5-hydroxy position ofserotonin MIC (μg/mL) Compound Bacterial Strain 1 7 8 9 10 11 12 E. coli6 >74 42 6.3 13 14 >124 BW25113 E. coli 1.5 >74 42 3.2 3.2 1.8 >124BW25113 ΔtolC Psuedomonas 24 >74 >83 >101 25 56 >124 aeruginosaSalmonella 6 >74 42 6.3 6.3 7 >124 typhimurium Vibrio 6 >74 42 6.3 6.37 >124 cholerae Shigella 6 >74 42 3.2 6.3 7 >124 boydii Klebsiella 6 >7442 6.3 13 7 >124 pneumoniae Enterobacter 6 >74 42 13 13 14 >124aerogenes Acinetobacter 6 >74 42 6.3 13 7 >124 baumannii Edwardsiella6 >74 42 25 25 28 >124 tarda Morganella 12 >74 >83 >101 >101 >112 >124morganii Staphylococcus 3 >74 42 6.3 6.3 3.5 >124 aureus

TABLE 5 Minimum inhibitory concentration (MIC) of various serotoninanalogs with structural modification other than the O-alkyl chain. MIC(μg/mL) Compound Number Bacterial Strain 2 3 4 5 6 E. coliBW25113 >51 >56 >61 >61 >61 E. coli BW25113 >51 >56 >61 >61 >61 ΔtolC

Based on the known biological activity of Compound 1, and without beingheld to theory, it was hypothesized that that these compounds may bemost useful for treating pathogens topically. The activity of Compounds1-3 and 7-11 against Staphylococcus aureus (3.0 μg/mL), Pseudomonasaeruginosa (24 μg/mL), and Streptococcus pyogenes (24 μg/mL) was tested.Utilizing Compound 1 as a topical therapeutic is attractive becausebioavailability and toxicity have not yet been published for humanclinical trials. For rodent studies, the current dosage of Compound 1 is10⁻⁸ to 10⁻⁴ mmol/kg, which is approximately 0.1 μM. Comparingtoxicological data of Compound 1 with the MIC measurements, topicalapplication would be a suitable method of dosing. However, in anotherembodiment, dosing would be systemic.

Example 4 Measurement of Minimum Inhibitory Concentrations forAdditional Analogs of Compound 1

Compounds 13, 14 and 15 were also tested in the bacterial MIC assay.

TABLE 6 Minimum inhibitory concentration (MIC) values of tryptamineanalogs with modification of the primary amine against E. coli BW25113,E. coli BW25113 ΔtolC, Bacillus subtilis 168 and a panel of clinicallyisolated pathogens. MIC (μg/mL) Compound Bacterial Strain 13 14 15 E.coli BW25113 >130 >83 52 E. coli BW25113 ΔtolC >130 >83 6.5 Bacillussubtilis 168 >130 >83 52 Psuedomonas aeruginosa >130 >83 52 Salmonellatyphimurium >130 >83 26 Vibrio cholerae >130 >83 >104 Shigellaboydii >130 >83 26 Klebsiella pneumoniae >130 >83 26 Enterobacteraerogenes >130 >83 52 Acinetobacter baumannii >130 >83 52 Edwardsiellatarda >130 >83 26 Morganella morganii >130 >83 >104 Staphylococcusaureus >130 >83 6.5

Removing or protecting the primary amine on the tryptamine of Compound 1abolished antibacterial activity, while replacement of the indole ringwith a benzene ring maintained activity.

The activity of a phenyl (benzene ring replacement of indole ring intryptamine) analog was also tested.

TABLE 7 Minimum inhibitory concentration (MIC) values of 1 and a phenyl(benzene ring replacement of indole ring in tryptamine) analog againstE. coli BW25113, E. coli BW25113 ΔtolC and a panel of clinicallyisolated pathogens. MIC (μg/mL) Compound Bacterial Strain 1 16 E. coliBW25113 20 5.3 E. coli BW25113 ΔtolC 5 2.6 Psuedomonas aeruginosa 24 21Salmonella typhimurium 6.0 5.3 Vibrio cholerae 6.0 5.3 Shigella boydii6.0 5.3 Klebsiella pneumoniae 6.0 5.3 Enterobacter aerogenes 6.0 5.3Acinetobacter baumannii 6.0 5.3 Edwardsiella tarda 6.0 5.3 Morganellamorganii 12 11 Staphylococcus aureus 3.0 5.3

Replacement of the indole ring with a benzene ring did not substantiallydiminish the antimicrobial activity.

In addition, the position and length of the ethylene moiety was changed.

TABLE 8 Minimum inhibitory concentration (MIC) values of 1 and phenylanalogs with position shift and length variation of the ethylene moiety.MIC (μg/mL) Compound Bacterial Strain 1 16 17 18 19 E. coli BW25113 65.3 5.3 9.4 >40 E. coli BW25113 ΔtolC 1.5 2.6 1.3 4.7 10 Bacillussubtilis 0.76 0.66 0.66 4.7 40

Shortening the ethylene moiety impaired the antimicrobial activity ofthe compounds.

Example 5 Rabbit RBC Hemolysis Assay

Prior to preparing the RBCs, we serially diluted compound-containing PBSsolutions into a 96-well plate (a final volume of 100 μL/well). A 0.25%Triton X solution was included as a positive control for hemolysis. Foreach assay, removed 1 mL of the RBC suspension was removed from thestock bottle and the cells were centrifuged for 2 min at 2000 rpm.Pelleted cells were resuspended in sterile PBS solution and centrifugedagain. Washing was repeated three times. The cells were resuspended inPBS and diluted 5-fold into the same solution. 100 μL aliquots of RBCswere added into wells of a 96-well plate that contained an equal volumeof a solution of compound in PBS. The plates were incubated for 6 or 17hours at 37° C. During the incubation, un-lysed RBCs settled at thebottom of the wells. At the end of the incubation, 90 μL of thesupernatant was transferred into the wells of a fresh 96-well plate, andmeasured the absorbance of the heme at λ=405 nm.

The results are shown in FIGS. 1 A and B. For all 6 compounds tested, atconcentrations higher than the MIC, hemolysis was high relative to thepositive control (Triton X) for both the 6 hour and 17 hour treatmenttimes. At the MIC and sub-MIC levels the hemolysis was lower,demonstrating some potential for toxicity improvement with syntheticdevelopment.

The use of the terms “a” and “an” and “the” and similar referents(especially in the context of the following claims) are to be construedto cover both the singular and the plural, unless otherwise indicatedherein or clearly contradicted by context. The terms first, second etc.as used herein are not meant to denote any particular ordering, butsimply for convenience to denote a plurality of, for example, layers.The terms “comprising”, “having”, “including”, and “containing” are tobe construed as open-ended terms (i.e., meaning “including, but notlimited to”) unless otherwise noted. Recitation of ranges of values aremerely intended to serve as a shorthand method of referring individuallyto each separate value falling within the range, unless otherwiseindicated herein, and each separate value is incorporated into thespecification as if it were individually recited herein. The endpointsof all ranges are included within the range and independentlycombinable. All methods described herein can be performed in a suitableorder unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”), is intended merely to better illustrate theinvention and does not pose a limitation on the scope of the inventionunless otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element as essential to thepractice of the invention as used herein.

While the invention has been described with reference to an exemplaryembodiment, it will be understood by those skilled in the art thatvarious changes may be made and equivalents may be substituted forelements thereof without departing from the scope of the invention. Inaddition, many modifications may be made to adapt a particular situationor material to the teachings of the invention without departing from theessential scope thereof. Therefore, it is intended that the inventionnot be limited to the particular embodiment disclosed as the best modecontemplated for carrying out this invention, but that the inventionwill include all embodiments falling within the scope of the appendedclaims. Any combination of the above-described elements in all possiblevariations thereof is encompassed by the invention unless otherwiseindicated herein or otherwise clearly contradicted by context.

The invention claimed is:
 1. An antimicrobial compound of formula 10,


2. A pharmaceutical composition comprising the antimicrobial compound ofclaim 1 and a pharmaceutically acceptable excipient.
 3. Thepharmaceutical formulation of claim 2, in the form of a composition fortopical administration.
 4. The pharmaceutical formulation of claim 2, inthe form of a composition for systemic administration.
 5. A method oftreating a subject in need of treatment for a bacterial infectioncomprising administering to the subject a compound of Formula II, or apharmaceutically acceptable salt thereof,

wherein R is a substituted or unsubstituted C₅-C₁₈ alkyl group, asubstituted or unsubstituted C₅-C₁₈ alkenyl group, a substituted orunsubstituted C₅-C₁₈ alkynyl group, a substituted or unsubstitutedC₅-C₁₈ cycloalkyl group, a substituted or unsubstituted C₆-C₁₈cycloalkenyl group, a substituted or unsubstituted C₈-C₁₈ cycloalkynylgroup, a substituted or unsubstituted C₅-C₁₈ heteroalkyl group, asubstituted or unsubstituted C₅-C₁₈ heterocycloalkyl group, asubstituted or unsubstituted C₇-C₃₀ arylalkyl group, a substituted orunsubstituted C₇-C₃₀ aryloxyalkyl group, or a substituted orunsubstituted C₇-C₃₀ arylthioalkyl group.
 6. The method of claim 5,wherein, R is a substituted or unsubstituted C₅-C₁₈ alkyl group, asubstituted or unsubstituted C₅-C₁₈ alkenyl group, or a substituted orunsubstituted C₅-C₁₈ alkynyl group.
 7. The method of claim 6, wherein Ris a substituted or unsubstituted C₅-C₁₈ alkyl group.
 8. The method ofclaim 7, wherein R is substituted with one or more C₁-C₂₀ alkyl groups,C₆-C₂₀ aryl, and/or C₇-C₁₃ arylalkyl groups.
 9. The method of claim 7,wherein R is substituted with one or more C₁-C₄ alkyl groups or a C₆-C₁₀aryl group.
 10. The method of claim 5, wherein the compound of FormulaII is


11. The method of claim 5, wherein the bacteria causing the infectionare Gram-negative bacteria, Gram-positive bacteria, or bacteria that areneither Gram-positive nor Gram-negative.
 12. The method of claim 11,wherein the Gram-negative bacteria is Escherichia coli, Pseudomonasaeruginosa, Candidatus liberibacter, Agrobacterium tumefaciens,Branhamella catarrhalis, Citrobacter diversus, Enterobacter aerogenes,Klebsiella pneumoniae, Proteus mirabilis, Salmonella typhimurium,Neisseria meningitidis, Serratia marcescens, Shigella sonnei, Shigellaboydii, Neisseria gonorrhoeae, Acinetobacter baumannii, Salmonellaenteriditis, Fusobacterium nucleatum, Veillonella parvula, Bacteroidesforsythus, Actinobacillus actinomycetemcomitans, Aggregatibacteractinomycetemcomitans, Porphyromonas gingivalis, Helicobacter pylori,Francisella tularensis, Yersinia pestis, Vibrio cholera, Shigellaboydii, Morganella morganii, Edwardsiella tarda, Campylobacter jejuni,or Haemophilus influenzae.
 13. The method of claim 11, wherein theGram-positive bacteria is a species of Bacillus, Listeria,Staphylococcus, Streptococcus, Enterococcus, Corynebacterium,Propionibacterium or Clostridium.
 14. The method of claim 13, whereinthe Gram-positive bacteria is Staphylococcus aureus, Staphylococcusepidermidis, Enterococcus faecium, Enterococcus faecalis, Streptococcuspyogenes, Bacillus cereus, or Bacillus anthracis.
 15. The method ofclaim 11, wherein the bacteria are carbapenam-resistant bacteria,methicillin-resistant Staphylococcus aureus, vanccomycin-resistantEnterococci or multi-drug resistant Neisseria gonorrhoeae.
 16. Themethod of claim 5, wherein the subject is a mammal.
 17. The method ofclaim 16, wherein the mammal is a human.
 18. The method of claim 5,wherein administration is topical administration.
 19. The method ofclaim 5, wherein administration is systemic administration.
 20. A methodof inhibiting bacterial growth comprising contacting bacteria with aneffective amount of the antimicrobial compound of Formula II or apharmaceutically acceptable salt thereof,

wherein R is a substituted or unsubstituted C₅-C₁₈ alkyl group, asubstituted or unsubstituted C₅-C₁₈ alkenyl group, a substituted orunsubstituted C₅-C₁₈ alkynyl group, a substituted or unsubstitutedC₅-C₁₈ cycloalkyl group, a substituted or unsubstituted C₆-C₁₈cycloalkenyl group, a substituted or unsubstituted C₈-C₁₈ cycloalkynylgroup, a substituted or unsubstituted C₅-C₁₈ heteroalkyl group, asubstituted or unsubstituted C₅-C₁₈ heterocycloalkyl group, asubstituted or unsubstituted C₇-C₃₀ arylalkyl group, a substituted orunsubstituted C₇-C₃₀ aryloxyalkyl group, or a substituted orunsubstituted C₇-C₃₀ arylthioalkyl group.
 21. The method of claim 20,wherein, R is a substituted or unsubstituted C₅-C₁₈ alkyl group, asubstituted or unsubstituted C₅-C₁₈ alkenyl group, or a substituted orunsubstituted C₅-C₁₈ alkynyl group.
 22. The method of claim 21, whereinR is a substituted or unsubstituted C₅-C₁₈ alkyl group.
 23. The methodof claim 20, wherein R is substituted with one or more C₁-C₂₀ alkylgroups, C₆-C₂₀ aryl, and/or C₇-C₁₃ arylalkyl groups.
 24. The method ofclaim 20, wherein R is substituted with one or more C₁-C₄ alkyl groupsor a C₆-C₁₀ aryl group.
 25. The method of claim 20, wherein the compoundof Formula II is


26. The method of claim 20, wherein the bacteria are actively growing orare in the stationary phase.
 27. The method of claim 20, wherein thebacteria are in the form of a biofilm.